US20230183780A1 - Dna probes for in situ hybridization on chromosomes - Google Patents

Dna probes for in situ hybridization on chromosomes Download PDF

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US20230183780A1
US20230183780A1 US16/320,325 US201616320325A US2023183780A1 US 20230183780 A1 US20230183780 A1 US 20230183780A1 US 201616320325 A US201616320325 A US 201616320325A US 2023183780 A1 US2023183780 A1 US 2023183780A1
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nucleic acid
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Wolfgang Weglöhner
Sabrina Schindler
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InVivo BioTech Services GmbH
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    • C12Q1/6841—In situ hybridisation
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    • C12Q2525/00—Reactions involving modified oligonucleotides, nucleic acids, or nucleotides
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    • C12Q2525/155—Modifications characterised by incorporating/generating a new priming site
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    • C12Q2565/00—Nucleic acid analysis characterised by mode or means of detection
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    • C12Q2600/156—Polymorphic or mutational markers
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • the invention relates to methods for producing probes for an in-situ hybridization of chromosomes for the diagnosis of chromosome aberrations and DNA probes and probe mixtures thus produced.
  • Chromosome aberrations can often be observed in tumor cells. They can be determined by G-banding or in-situ hybridization (ISH) with probes for specific genome loci. In multicolor fluorescence in-situ hybridization, several regions of the genome are labeled in different colors; these regions can also be located at a distance from the loci of diseases and breakpoints, so that even complex chromosome aberrations can easily be diagnosed. Some aberrations occur only with specific tumors, for example the Philadelphia chromosome with certain leukemias; others indicate the type of medical treatment, particularly with tumors of the breast. Clinically relevant here are the oncogene ERBB2, which codes for a cell surface receptor, and the CEN17 region for the centromere region of chromosome 17. ISH then provides a means of assessing the aggressiveness of the tumor and a more targeted treatment for the patients. Subgroups of non-Hodgkin's lymphomas can also be distinguished via genetic variations.
  • ISH probes Conventional methods of producing ISH probes use BAC clones (Bacterial Artificial Chromosome), YAC clones, cosmids and fosmids. These contain large regions of genomic DNA (up to 500 kilobases) and thus repetitive sequences, pseudogenes and paralogous sequences in addition to the sequences sought. They cause nonspecific hybridizations and background in chromogenic or fluorescence ISH, which makes an evaluation more difficult and sometimes impossible. In CISH (Chromogenic In-Situ Hybridization) in particular, a high background is often unavoidable. CISH probes are usually smaller than FISH (Fluorescence In-Situ Hybridization) probes.
  • FISH Fluorescence In-Situ Hybridization
  • the BAC clones are randomly fragmented, and the fragments are equipped with linkers and amplified in a PCR.
  • the DNA is dissolved and digested at 65° C. with a double-strand specific nuclease (DSN—duplex-specific nuclease).
  • DSN double-strand specific nuclease
  • the amplification and digest steps in the presence of Cot-1DNA are repeated several times, until a probe or a probe mixture “free” from repetitive human sequences is obtained.
  • the removal of the repetitive sequences can also be achieved by other methods; see Craid J M et al, Removal of repetitive sequences from FISH probes using PCR - assisted affinity chromatography, HUMAN GENETICS (Springer, Berlin, DE) Vol. 100, pp. 472-476; US 2004029298, WO 2004/083386; WO 01/06014. But these methods not only involve considerable effort, they are unfortunately also never complete or certain. Neither is it possible to simply secure the probe DNA in
  • ISH probes can also be produced in a PCR (polymerase chain reaction).
  • the PCR takes place on the genome, and specifically on regions which are free from repetitive sequences, and often code for only part of a gene or a domain. Repetitive and Alu sequences can be found everywhere in the human genome and on all chromosomes.
  • U.S. Pat. No. 8,407,013 B2 discloses a computer-assisted sequence analysis and an ab-initio generation of genomic probes by PCR; see Rogan P K et al Sequence - based design of single - copy genomic DNA probes for fluorescence in situ hybridization, Genome Res. (2001) 11(6): 1086-1094.
  • the probe must, furthermore, additionally be labeled with radioactive, chromogenic or fluorescent groups.
  • the labeling can be done enzymatically by means of a nick translation reaction, by random priming, or by direct PCR labeling with labeled nucleotides and/or by chemical coupling.
  • the labeling in the amplification reaction is complex, however, because the polymerase chain reaction has to be re-established for every labeling, every fluorescent dye, and every chromogenic group. It also depends on sequence length, chemical structure of the modification, length of the linker between labeling and nucleotide, and also on the polymerase and the condition of the starting material.
  • the Prior Art thus represents a problem.
  • the method comprises the production of directly or indirectly labeled nucleic acids, comprising an analysis of sequences in larger genomic regions for segments with specific sequences and the selection of specific nucleic acid sequences for specific loci; design and synthesis of sense and antisense primer pairs for a polymerase chain reaction on selected, specific nucleic acid sequences, where the synthesized primers each contain a sequence which is complementary to the strand or complementary strand of the non-specific nucleic sequence, and a non-complementary uniform linker sequence, which does not hybridize with the genome under stringent conditions and can facultatively contain a cleavage sequence for a restriction endonuclease; a number of first polymerase chain reactions with the number of sense and antisense primer pairs and, after combining the reaction products, obtaining a first mixture (pool A) of synthesized PCR fragments which contain known (non-repetitive) sequences; a multiplex polymerase chain reaction on the mixture (pool A) of synthesized PCR fragments with the aid of
  • the synthesized nucleic acid fragments which are present in the mixture after the first polymerase chain reaction are analyzed size-selectively and then purified. Furthermore, it is advantageous to add modified or labeled nucleotides (PCR labeling) in the last amplification step. If nucleotides which have been modified in the last amplification step are added, they can be of a type which allows chemical coupling with a chromogenic or fluorescent group, preferably aminoallyl NTPs.
  • the nucleic acid fragments which result from the first polymerase chain reaction are cloned in plasmids.
  • the specialist will recognize that this can be done under restriction into the linker sequence. After amplification of the plasmids, the fragments can be generated in any quantity via the linker.
  • Probe fragments can also be subjected to a reaction which inserts or attaches reporter groups into/onto the hybridization probe.
  • the labels inserted can be radioactive, chromophoric or fluorescent.
  • the chromophoric group includes haptens such as biotin, avidin, digoxigenin, because these haptens can be made visible in an immunoreaction with a labeled antibody in the known way. Further chromophoric groups are enzymes such as peroxidases or lactases, which catalyze a color reaction. It is also possible to use modified nucleotides with a reactive group such as allylamine, which can be subjected to a reaction with appropriate groups of dyes.
  • the method disclosed has the advantage that the non-repetitive nucleic acid sequences selected in step (a) can be selected such that they are amplified in the first multiple polymerase chain reaction with essentially the same frequency.
  • the sequence segments are preferably selected in step (a) such that non-repetitive PCR fragments with 100 to 5,000 base pairs, preferably with 100 to 1,000 base pairs, result. Particularly preferred are fragments with 400 to 600 base pairs.
  • the non-repetitive nucleic acid sequences which are selected in the analytical step are adjacent to one another on the genome under analysis so that a higher signal intensity results from the in-situ hybridization.
  • probes with different labeling are often required to detect chromosome aberrations.
  • the disclosure therefore also encompasses the production of a large number of probes with different labels. It is advantageous if the specific nucleotide sequences selected in the first step are adjacent to a breakpoint region in the chromosome. Particularly advantageous and practical for diagnostic purposes is when the specific sequences selected in the analytical step flank a breakpoint region and have different labels, since a chromosome aberration can thus be diagnosed directly.
  • the different labels of the probes can also be selected for adjacent sequences such that the color stains initially result in a compound color, and a color change or two different color signals can be observed in the case of an aberration. The reverse process can also take place, i.e.
  • two different color signals can produce a compound signal or a fusion signal if there is an aberration.
  • sequences which are from a single region, or which flank this region, which is amplified in the case of an aberration, if required as part of a balanced, unbalanced and reciprocal translocation.
  • the labeling of the probes is preferably selected from the group: chromogenic molecules, polymethine dyes, thiazole and oxazole dyes, Hoechst 33342 (2′-(4-ethoxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5′-bi-1H-benzimidazole trihydrochloride), 4′,6-diamidin-2-phenylindole, Alexa 405, Alexa 488, Alexa 594, Alexa 633; Texas Red, rhodamine; sulfonated and non-sulfonated cyanine dyes, Cy2, Cy3, Cy5, Cy7; fluorescent molecules, fluorescein, 5,6-carbofluorescin, FITC (fluorescein isothiocyanate), GFP (Green Fluorescent Protein); chemiluminescent molecules, acridinium; ATTO®-fluorescent dyes (Atto-Tec, Siegen, DE), PromoFluor
  • One embodiment relates to the provision of a labeled probe for an in-situ hybridization to detect a chromosome aberration, comprising a plurality of PCR fragments whose sequences do not contain any repeats, pseudogenes or paralogous genes, and which are adjacent to each other on the human genome.
  • a further embodiment relates to a probe mixture or a detection kit for a specific chromosome aberration which contains several differently labeled probes which flank the particular breakpoint regions.
  • FIG. 1 A diagram of the steps to produce sequence-controlled PCR probes for the in-situ hybridization (ISH) of chromosomes;
  • FIG. 2 A schematic diagram of the steps to obtain repeat-reduced ISH probes according to the Prior Art
  • FIG. 3 Fluorescence microscopy images of samples after control staining of the cell nucleus with DAPI (4′,6-diaminophenolindol) and various in-situ hybridizations, where the sequence-controlled ISH probe was labeled in the PCR reaction (“one-step”): left column: control staining of the cell nucleus with DAPI; right column: in-situ hybridization with probes for the HER2, MDM2, MET and FGFR1 genes or the centromere;
  • FIG. 4 Fluorescence microscopy images of samples after control staining of the cell nucleus with DAPI (4′,6-diaminophenolindol) and various in-situ hybridizations, where the sequence-controlled ISH probe was labeled by means of nick translation: left column: control staining of the cell nucleus with DAPI; right column: in-situ hybridization with probes for the HER2, MDM2, MET and FGFR1 genes or the centromere;
  • Sequence-controlled PCR probes can be generated from genomic DNA.
  • the complete sequence of the human genome is known and can be obtained from databases. It is the starting point for the design of FISH/CISH probes. It is also possible to start with the sequence of BAC clones or other DNA carriers (plasmids, cosmids, fosmids, YACs) if the carriers and their sequences are available. Everything described below for genomic DNA can also be conducted with the other carriers.
  • a first step (a) the genomic sequence and the genetic composition of the region is investigated by means of a computer analysis using the sequences in the databases.
  • all regions with simple repeats, Alu sequences and complex repeats as well as all pseudogenes and paralogous sections are sought and identified in the genomic sequence. These sequences are unsuitable for the ISH probe.
  • the NCBI National Center for Biotechnology Information, Bethesda, Maryland
  • ENSEMBL maintained by the European Bioinformatics Institute (EBI) and the European Molecular Biology Laboratory (EMBL), Heidelberg, DE
  • EBI European Bioinformatics Institute
  • EMBL European Molecular Biology Laboratory
  • Any nonspecific sequence regions can be further narrowed down and identified with the aid of conventional analytical programs.
  • step a Only sequences which are specific to a locus and occur once are included in the hybridization probe. Excluded are sequences which hybridize at other loci also during a chromosome hybridization and thus contribute nonspecifically to the background (step a).
  • a library of sense and antisense primers is then collated (b). Each primer is synthesized with a specific primer sequence and a uniform linker. Sense and antisense primers are planned and selected on the basis of the known genome sequence so that the PCR and the subsequent amplification result in DNA fragments of essentially the same length.
  • 200 primers are designed and synthesized for every genome region so that, after the PCR, the locus on the genome is covered section-by-section by 100 DNA fragments.
  • the specific fragments are generated in individual PCR reactions with the aid of the primers; the resulting PCR fragments are checked for purity and size and combined or pooled (pool A) in step (d).
  • 10 ng of pooled PCR-DNA is used as the matrix for a second amplification—(step e)—where the linkers serve as sense and antisense primers in this amplification.
  • pool B with labeled PCR fragments. After purification, these can be used in the in-situ hybridization. Alternatively, pool B containing the mixed PCR fragments can also be labeled by nick translation after the second amplification.
  • dNTPs labeled with fluorescent dyes (for example Atto488 or Cy3) or with haptens (for example digoxigenin, dinitrophenol or biotin), where the labeling can be done directly in the linker PCR.
  • fluorescent dyes for example Atto488 or Cy3
  • haptens for example digoxigenin, dinitrophenol or biotin
  • this ratio will be between 1:1 and 1:20; or (c) chemically reactive dNTPs, such as aminoallyl dNTP.
  • HER2 On chromosome 17, the genome region with the HER2 gene and large 5′ and 3′ flanks was selected for the design of the HER2 (ERBB2) gene probe.
  • the HER2 gene probe covered the section from 39,395,605 to 39,799,506 on chromosome 17.
  • the genome sequence was analyzed in the Ensembl (www.ensembl.org) and NCBI (https://www.ncbi.nlm.nih.gov/) databases, and four suitable sections were identified for the probe.
  • the first genome section was chromosome 17: 39,395,605-39,569,361. The explanation below refers only to this section; the three other genomic sections at the HER2 locus were processed analogously.
  • Sense and antisense primers for the specific sequence sections were planned and synthesized for genome fragments with 250 to 800 base pairs.
  • the primers were complementary to the genome sequence which was to be amplified; on the other hand, they also contained a universal linker sequence with a cleavage site for a restriction endonuclease.
  • Sense and antisense primers were planned such that the resulting products in the PCR on the genome were of similar length. The desired fragment length was approx. 500 base pairs. The only deviation from this was when the specificity or the functionality of the primer pair required it.
  • Approx. 200 primers were designed and synthesized for each of the four genomic sections. Table 1, which is appended to the description, contains a representative list of the thus determined sense and antisense primers for the genome section 39,395,605-39,569.361 on chromosome 17.
  • the genome fragments were amplified in 50 ⁇ l solutions for every sense and antisense primer pair.
  • the individual PCR reactions were conducted in the high-throughput method (96-well) at an attachment temperature (primer annealing) of 55° C. and 15 seconds strand elongation over 35 cycles in each case.
  • the PCR fragments obtained were checked for purity and size on the agarose gel. Only PCR products with a specific band of expected size were used so that their sequence was effectively controlled and known. The yield of correct PCR products was over 90%.
  • PCR products were usually blunt cloned into a carrier plasmid without cutting and thus secured. Some PCR products were also cloned into a plasmid after cutting. The occasional check of the sequence correctness was unproblematic and conducted in the known way.
  • PCR products were pooled and processed to remove primers, proteins, free nucleotides and salts.
  • the second amplification via the universal linkers was done with a high-performance Taq polymerase for large yields, directly with the mixture of the individual fragments (pool A) as the template (multiplex PCR).
  • dNTPs labeled with fluorescent dyes e.g. Atto488 or Cy3 were used in this multiplex PCR/linker PCR.
  • the fluorescence-labeled dNTPs were added in a tested ratio which was dependent on the labeled dNTP and differed according to the type of probe (gene probe or centromere probe). The ratio was between 5:1 and 1:5 across the different probes and depending on the labeling.
  • the labeling and amplification reaction was followed by a final purification of the labeled multiplex PCR probes by precipitation and chromatography, where free dNTPs, labeled nucleotides, sense and antisense primers as well as the enzyme were removed. This was followed by a photometric measurement and determination of the insertion rate of the fluorescence in the FISH probes as well as a final check of the probe by agarose gel electrophoresis.
  • the DNA probes were post-fragmented if they produced too much background in the ISH because of their length. In general, a length of 200 to 300 base pairs is favorable for ISH.
  • the post-fragmentation was done physically, but can also be done enzymatically or chemically.
  • the fragment size distribution was analyzed by agarose gel electrophoresis.
  • the HER2 locus probe comprised four segments.
  • the above-mentioned steps 1 to 8 concerned a first section.
  • the final DNA probe consisted of four separate preparations (4 multiplex PCR probes).
  • these four individual preparations can be combined after the universal linker amplification, and labeled and purified together, although this affords less control.
  • probe type amplification probe, break-apart probe, fusion probe
  • a DNA probe mixture will consist of 1 to 10 preparations (multiplex PCR probes).
  • ISH with DNA probes can be conducted using standard methods on tissues or cells, for example in accordance with the recommendations of the Laboratory Working Group of the DGHO (German Society for Hematology and Medical Oncology). In this case, in-situ hybridization takes place on interphase cells of cell cultures or tissues.
  • the target DNA in each case is the nuclear DNA of interphase cell nuclei, which are fixed on a specimen slide.
  • the probe here is produced and labeled as described in Example 1.
  • the cell nuclei are typically counterstained with the fluorochrome DAPI (4′,6-diamidino-2-phenylindole).
  • FISH can be conducted on the following materials: peripheral blood (PB), bone marrow (KM), paraffin sections, tumor tissue, cytospin preparations, amniotic fluid, cells/metaphases fixed with methanol glacial acetic acid, etc.
  • PB peripheral blood
  • KM bone marrow
  • paraffin sections tumor tissue
  • cytospin preparations amniotic fluid
  • cells/metaphases fixed with methanol glacial acetic acid etc.
  • Other patient samples such as blood or bone marrow are fixed with methanol/ethanoic acid (ratio 3:1) after Ficoll separation and frozen at ⁇ 20° C. until hybridization.
  • Special pretreatments are necessary for amniotic fluid or paraffin sections.
  • FIGS. 3 and 4 depict the results for differently labeled probes (by nick translation or one-step-PCR labeling). In both cases, the hybridization signals were clearly visible and the background hybridization was negligible.

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DE102016124844.4A DE102016124844B3 (de) 2016-07-25 2016-12-19 DNA-Sonden für eine In-Situ-Hybridisierung von Chromosomen
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CN111979297B (zh) * 2019-05-22 2023-10-27 南京农业大学 基于多重pcr合成寡核苷酸探针的方法
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KR102429576B1 (ko) 2020-04-29 2022-08-03 경희대학교 산학협력단 전도성 태그를 이용한 dna 염기서열을 식별하는 방법 및 장치
CN112795649A (zh) * 2021-01-05 2021-05-14 武汉友芝友医疗科技股份有限公司 一种用于检测her2基因扩增水平的探针组及其应用
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